Multiple amplification cycle detection

A method for analyzing a target nucleic acid in a sample comprising

• (a) providing a sample container comprising a first blister and a second sample well, the second sample well comprising therein primers for amplifying the target nucleic acid,
• (b) subjecting the sample to a first amplification in the first blister, and moving a portion of the sample from the first blister to the second sample well,
• (c) subjecting the second sample well to amplification conditions through a plurality of amplification cycles to amplify the target nucleic acid in the second sample well,
• (d) generating a melt curve of the amplified target nucleic acid in the second sample well,
• (e) repeating steps (c) and (d), and
• (f) determining a Cp by identifying the amplification cycle in which a value for the melt curve exceeds a predetermined value,
• wherein the sample in the second sample well contains two or more different target nucleic acids and the second sample well contains primers for amplifying the two or more different target nucleic acids, and the method further comprises:
• simultaneously amplifying the two or more target nucleic acids in the second sample well through a plurality of amplification cycles,
• generating a plurality of melt curves of the amplified two or more target nucleic acids,
• determining a value for each of the plurality of melt curves from each of the amplified two or more target nucleic acids, and
• determining a melt Cp for each of the two or more target nucleic acids by identifying a melt event in which the value for the melt curve for each of the two or more target nucleic acids exceeds a predetermined value.